Method for measuring dna polymerization and applications of the method

ABSTRACT

A method for measuring DNA-dependent DNA polymerisation in a biological sample, is described. The method comprises the steps of providing a primer with a single stranded short specific sequence, which is unable to base pair internally, bound to a solid phase; contacting the primer construct with a reaction mixture containing a single stranded deoxynucleotide template with a part of the sequence complementary to the primer and the four deoxynucleoside triphosphates, one of which is modified so that it is specifically recognized by a labeled antibody; adding a biological sample comprising the DNA polymerase, such as retrovirus reverse transcriptase (RT), to the mixture; allowing the polymerase reaction to proceed; incubating the immobilized reaction product with the labeled antibody; detecting the amount of bound labeled antibody; and measuring the amount of incorporated modified deoxynucleoside triphosphate, as a measure of the DNA polymerisation, which may be used drug susceptibility testing. A commercial package is also disclosed.

[0001] The present invention relates to a method for measuring DNApolymerization and applications of the method. More precisely, theinvention relates to a method for measuring DNA-dependent DNApolymerization.

BACKGROUND

[0002] The latest decades have experienced a rapid development of newmethods for measurement of reverse transcription, RNA dependent DNApolymerization. The more complex issue concerning methods forquantification of DNA dependent DNA polymerization has so far receivedmuch less attention.

[0003] The classic DNA polymerase activity assays involve use of DNAsetreated DNA (“activated DNA”) as primer/template and incorporation ofradiolabeled nucleotides into DNA (Aposhian and Komberg 1962).Measurement of acid preciptable radioactivity allows calculation of theamount of nucleotides incorporated and the number of enzyme unitspresent. However, use of radioactivity is currently restricted anddiscouraged in many laboratories and there is due to this a generaltrend away from radioactivity-based techniques.

[0004] For DNA polymerases a commercial assay based on ELISA detectionof digoxigenine labeled nucleotides incorporated in newly made DNA isavailable (Roche Molecular Biochemicals Cat. no 1468120, U.S. Pat. No.5,635,350). This assay is hampered by the use of two differentnucleotide substrate analogues with bulky groups, digoxigenine as labeland biotin for product immobilization. As a result the polymerizationreaction velocity and subsequent detection sensitivity is reduced. Theutilization of substrate analogues with highly deviating kineticproperties makes this system less relevant for studies of drugsusceptibility of different polymerases.

[0005] Another more attractive alternative method is afluorescence-based assay for DNA polymerase holoenzyme, based on thespecific reaction of the dye PicoGreen with double-stranded DNA (Sevilleet all 1996). The latter process has recently been modified to make itsuitable for a broader range of different DNA polymerizing enzymes(Tveit and Kristensen 2001). This assay is technically simple and basedon the utilization of natural nucleotides. The detection sensitivity is,however, still in the same range as the classic radioactive DNApolymerase assay and the applications described demonstrates a detectionrange of 0.05-0.5 U DNA polymerase/sample.

[0006] HIV therapy today is based on multidrug therapy. The regimens arebased on combinations of all three types of drugs available: nucleosideanalogues, non-nucleoside analogues and protease inhibitors. Thestrategy is to minimize the probability for a mutant virus to survive.

[0007] The reverse transcriptase (RT) inhibitors are either nucleosideanalogues or non-nucleoside analogues. The non-nucleoside inhibitorsbind to a hydrophobic pocket in the RT enzyme close to, but notcontiguous with, the active site. HIV-1 replication is inhibitedallosterically by displacing the catalytic aspartate residues relativeto the polymerase binding site.

[0008] The nucleoside inhibitors used today terminates the DNA chainelongation as they lack a 3′-hydroxyl group. Prolonged therapy withnucleoside inhibitors commonly leads to the development of resistantvirus. This process is associated with the gradual appearance ofmutations in the virus pol gene, each leading to defined amino acidsubstitutions (for a review see Vandamme et al 1998). The effects ofthese substitutions at the enzymatic levels is complicated and includesenhancement of a primitive DNA editing function. This reaction isnucleotide dependent and produces dinucleoside polyphosphate and anextendible DNA 3′ end (Arion et al 1998, Meyer et al 1999).

[0009] The HIV-1 RT as well as other reverse transcriptases performthree different enzymatic reactions: RNA-dependent DNA polymerization,DNA-dependent DNA polymerization, and degradation of RNA in the DNA-RNAhybrid (RNase H). The HIV reverse transcriptase, encoded by the polgene, is a heterodimer consisting of a p66 and a p51 subunit. BothRNA-dependent DNA polymerization and DNA-dependent DNA polymerizationare performed by the same active site localized in the p66 subunit (fora review see Goff 1990). The reaction mechanism of these drugs hasmainly been defined according to their action on the RNA-dependent DNApolymerization reaction. The effect on the DNA-dependent DNApolymerization reaction is comparatively less studied.

[0010] Provided that the reaction mechanism and the active metabolizeddrug is known and available, phenotypic virus drug susceptibility couldbe determined at the enzyme level. Depending on the capacity of theenzyme assays and the virus isolation techniques used, the drugsensitivity testing can theoretically be done either on supernatantsfrom virus culture propagation, the primary virus isolation or on viruspreparations recovered directly from the patients. Conventional RTactivity assay is performed by utilizing an artificial template-primerconstruction and labeled deoxynucleoside triphosphate as nucleotidesubstrate. The template/primer pair poly(rA)/oligo(dT) is the mostefficient and most used combination for determination of HIV as well asfor other retroviral RTs. A drawback of this type of assay when drugsensitivity testing is concerned, is that only non-nucleoside analoguesor analogues that can base pair with rA can be tested. Analogues to theother nucleotide bases will require an assay based on a variable polymertemplate. RNA polymers containing pyrimidine bases are notoriouslysensitive to RNases and in practice not compatible with biologicalsamples. It would therefore be advantageous to base a polymerase assayintended for drug sensitivity testing on a variable DNA template,provided that the assay system gives results that correlate with thosefrom inhibition of reverse transcription and classic phenotypic drugresistance tests.

[0011] HIV therapy used today is only one example of the potency of DNApolymerase inhibitors. The current situation concerning resistancedevelopment among bacteria and other microorganisms motivates the searchfor new classes of antimicrobial drugs. DNA polymerases are one of themajor targets during this effort. As such there is a great demand fortechnically simple polymerase assays, which do not cause potentialenvironmental hazards and can be applied for drug screening towards abroad range of microbial DNA polymerase isozymes. The toxicity of thedrug leads found must further be evaluated towards the correspondingmammalian DNA polymerases.

[0012] Quantification of proliferation associated polymerases such aspolymerase-α and -δ can be used for monitoring cell proliferation. Itmay be mentioned in this context that the serum levels of thymidinekinase, another cell proliferation associated enzyme, are currently usedfor prognosis and classification of malignant disease (U.S. Pat. No.4,637,977). Phosphorylation of thymidine is just one of the twointracellular synthetic pathways, which provides thymidine triphosphatefor DNA synthesis. Measurement of the DNA polymerase itself has thepotential to give a more correct estimation of total DNA synthesiscompared to thymidine kinase activity or thymidine incorporation.

DESCRIPTION OF THE INVENTION

[0013] The present invention provides a non-radioactive DNA polymeraseassay in microtiter plate format enabling colorimetric or fluorimetricproduct detection.

[0014] In a preferred embodiment it utilizes, as nucleoside triphosphatesubstrate, 5-bromodeoxyuridine 5′-triphosphate (BrdUTP). The differencein Van der Waals' radius between the 5′ position bromine in BrdUTP andthe 5′ position methyl group in thymidine triphosphate is minimal (1.95A compared to 2.0 A) and the enzyme kinetic properties of these twonucleotides are quite similar. The method can be aromatized and have adetection range down to 3 nU polymerase activity/sample.

[0015] One of the applications of the present invention is drugsusceptibility testing. All anti-retroviral drugs approved hithertointerfere with the enzymatic reaction of either the viral protease orthe RT. There are in addition candidate drugs in the pipeline whichaffect the function of the retroviral integrase.

[0016] In particular, the present invention provides a procedure tomeasure a broad range of different DNA-dependent DNA polymerases. It hasproved suitable even for studies of highly processive DNA polymerasesystems in spite of the comparatively short template used. Theusefulness for determination of the activity of bacterial polymerase Iand III, mammalian DNA polymerase α, β and γ, a proliferation associatedpolymerase activity in human serum and DNA dependent DNA polymerizationby HIV RT is demonstrated, but the method can be used for studies ofvirtually all viral and cellular DNA polymerases. One of the featuresthat distinguish the present. DNA polymerase assay from previous art isits outstanding sensitivity, which makes detection of down to 3 nU E.coli DNA polymerase I activity feasible.

[0017] Thus, one aspect of the invention is directed to a method formeasuring DNA dependent DNA polymerisation in a biological sample,comprising the steps of

[0018] a) providing a primer with a single stranded short specificsequence, which is unable to base pair internally, bound to a solidphase,

[0019] b) contacting the primer construct with a reaction mixturecontaining a single stranded deoxy nucleotide template with a part ofthe sequence complementary to the primer and the four deoxy nucleosidetriphosphates, one of which is modified so that it is specificallyrecognized by a labeled antibody,

[0020] c) adding a biological sample comprising the DNA polymerase tothe mixture of b),

[0021] d) allowing the polymerase reaction to proceed,

[0022] e) incubating the immobilized reaction product resulting from d)with the labeled antibody,

[0023] f) detecting the amount of bound labeled antibody with the aid ofthe label used, and

[0024] g) measuring the amount of incorporated modified deoxynucleotide,as a measure of the DNA polymerization, with the aid of the label of thebound antibody.

[0025] In an embodiment the DNA polymerization is made by a retrovirusreverse transcriptase (RT), such as human immunodeficiency virus (HIV)RT.

[0026] In another embodiment the modified deoxy nucleoside triphosphateis 5-bromodeoxyuridine 5′-triphosphate (BrdUTP) and the labeled antibodyis an alkaline phosphatase (Ap) conjugated anti-BrdU monoclonalantibody.

[0027] In a preferred embodiment of the method according to theinvention, the measured DNA polymerisation is used for drugsusceptibility testing.

[0028] The drug susceptibility testing is performed to evaluate if acertain drug is effective in a mammalian individual, and the result maybe used for selecting drug treatment therapy for that individual. Inpractice, the individual will be subjected to testing at several pointsof time to monitor the development of drug treatment in said individual.

[0029] The invention is also directed to a commercial package comprisingwritten and/or data carrier instructions for measuring DNA-dependent DNApolymerisation according the invention. The package will comprise atleast the following items:

[0030] a) a primer with a single stranded short specific sequence, whichis unable to base pair internally, bound to a solid phase,

[0031] b) a single stranded deoxynucleotide template with a part of thesequence complementary to the primer in a),

[0032] c) the four deoxynucleoside triphosphates, one of which ismodified so that it is specifically recognized by a labeled antibody,and

[0033] d) the labelled antibody that recognizes the modifieddeoxynucleoside triphosphate in c).

[0034] The invention will now be illustrated by the following unlimitingdescription of embodiments and drawings of the invention.

[0035] The teachings of the cited literature is incorporated herein byreference.

SHORT DESCRIPTIONS OF THE DRAWINGS

[0036]FIG. 1. demonstrates the effects of variation in template sequenceon inhibition of DNA polymerase III with TMAU.

[0037]FIG. 2. exemplifies the detection sensitivity of the DNApolymerase assay. HIV-1 wild type RT (♦), mammalian DNA polymerase β ()and E. coli DNA polymerase I (▴).

[0038]FIG. 3. demonstrates the ability of the DNA polymerase assay tomeasure inhibition by the dideoxy analogues to all the four DNA bases.Symbols: ddATP (▪), ddGT (♦), ddCTP () and ddTP (▴).

[0039]FIG. 4. shows that the biochemical mechanism underlying resistanceto the antiviral drug tenofovir is based on enhancement of an ATPdependent phosphorolysis reaction. Symbols: HIV-1 wild type RT instandard reaction solution (◯). HIV-1 wild type RT in reaction solutionwith ATP (), HIV-1 mutant RT in standard reaction solution (⋄), HIV-1mutant RT in reaction solution with ATP(♦).

[0040]FIG. 5. exemplifies determination of susceptibility to theantiviral drug Nevirapine using RTs isolated from plasma from HIVinfected individuals. Symbols: (□), (▪) and (▴) RTs from infectedindividuals () a control consisting of recombinant HIV-1 wild type RT,(♦) a control consisting of mutated (L100I) recombinant HIV-1 RT withintermediate Nevirapine resistance.

DESCRIPTION OF EMBODIMENTS

[0041] Production of Primer Coated Microtiter Plates

[0042] 1-ethyl-3-(3-dimethylamino-propyl)carboimide hydrochloride (finalconcentration 10 mg/ml) was added to a 100 mM 1-Methylimidazole buffer(pH 7.0) and the mixture was used to dilute the primer construct to afinal concentration of 1 μg/ml. 100 μl of the primer solution wasaliquoted to each well of a microtiter plate consisting of Nalge NuncNucleoLink® transparent strips (Cat no 248259). The plates wereincubated 6-8 hours at 37° C., washed thoroughly in 2M NaOH with 2 mMEthylenediaminetetraacetic acid (EDTA) and soaked in three 5 L vialswith water. Residual fluid in the wells was removed by tapping theplates upside down on absorbing cloth or paper. The plates were allowedto dry for 30 min at room temperature and finally frozen for storage at−20° C.

[0043] Protocol for DNA-Polymerase Assay.

[0044] The DNA-polymerase assay is based on a short primer with aspecific sequence that is covalently bound to the wells of a 96 wellmicrotiter plate. The reaction mixture contains a single strandeddeoxynucleotide template with a part of the sequence complementary tothe primer and the four deoxynucleoside triphosphates. Thymidinetriphosphate is, however, replaced by 5-bromodeoxyuridine5′-triphosphate (BrdUTP). The amount of bromodeoxyuridine monophosphate(BrdUMP) incorporated into DNA during the polymerase reaction, isdetected with an alkaline phosphatase (Ap) conjugated anti-BrdUmonoclonal antibody. An Ap substrate, 4-methylumbelliferyl phosphate, isused for fluorimetric product detection.

[0045] 100 μl of DNA polymerase reaction mixture was added to each wellof the primer coated microtiter plates. The samples were diluted in DNApolymerase base buffer and the polymerase reaction was initiated bytransferring 50 μl sample dilution to each well on the plate. Themicrotiter plate was incubated at 33° C. and reaction was terminatedafter indicated times by washing the plate in 3 mM borate buffer (pH8.9) with 1.5% (v/v)octophenoxypolyethoxyethanol (Triton X-100). Usuallytwo incubation times, 4 hours and over night (16 hours), were used tocheck the linearity of the polymerization reaction. The plates werewashed thoroughly in 2M NaOH with 2 mM EDTA and soaked in three 5 Lvials with water.

[0046] Next the plates were incubated for 90 minutes at 33° C. with 100μl alkaline phosphatase (Ap) conjugated anti-BrdU monoclonal antibodydiluted to 4.8 μg/ml in 25 mM(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl]-1,3-propanediol) (Bis Tris)buffer (pH 7.2) with 50 mM NaCl, 37.5 mM (NH₄)₂SO₄, 1 mg/ml Dextransulphate, 1% TritonX-100 and 25 mg/ml Sigma non-fat dried milk.

[0047] The plates were thereafter washed again in 3 mM borate buffer (pH8.9) with 1.5% (v/v) TritonX-100 to remove unbound labeled antibody. Thealkaline phosphatase activity was determined using 4-methylumbelliferylphosphate substrate dissolved in Tris-buffer (pH 8.9). Fluorescence wasread at 460 nm with a Wallac Victor 2 reader at defined intervals(excitation 355 mm).

[0048] Protocol for Determination of Inhibition of Second StrandSynthesis on Variable DNA Template.

[0049] The inhibition studies were performed in a modified DNApolymerase assay. The drugs were serially diluted in five steps and 25μl aliquots were transferred to each well in the microtiter plate, mixedwith 100 μl DNA polymerase reaction mixture and the enzyme reaction wasinitiated by addition of 25 μl enzyme dilution. Non-nucleoside analogueswere studied at standard reaction conditions while the concentration ofall four deoxynucleoside triphosphates (dNTP) were reduced to 1 μM inthe studies of dNTP competing inhibitors. The polymerase reaction wasallowed to proceed over-night (16-24 hours at 33° C.). Thereafter thereaction was terminated by a wash of the plate. The IC₅₀ value wasdefined as the concentration of drug giving 50% inhibition of thepolymerase activity studied.

[0050] Protocol for Determination of RT Activity.

[0051] A modification of the calorimetric RT assay (Cavidi® Lenti RTactivity kit), available from Cavidi Tech, Uppsala, Sweden was used forthe determination of the level of RT activity in the virus preparationsstudied. The method has been described (Ekstrand at al 1996). In short,poly(rA) covalently bound to the wells of a 96 well microtiter plateserves as template for the incorporation of 5-bromodeoxyuridine5′-triphosphate (BrdUTP) during the reverse transcription step at 33° C.The amount of bromodeoxyuridine monophosphate (BrdUMP) incorporated intoDNA, is detected with an alkaline phosphatase (Ap) conjugated anti-BrdUmonoclonal antibody. An Ap substrate, 4-methylumbelliferyl phosphate, isfinally used for fluorimetric detection.

[0052] Protocol for Determination of Inhibition of ReverseTranscription.

[0053] The inhibition studies were performed in a modified Cavidi HS-kitLenti RT assay. The inhibitors were serially diluted in five steps and25 μl aliquots were transferred to each well in the microtiter plate,mixed with 100 μl RT reaction mixture and the enzyme reaction wasinitiated by addition of 50 μl enzyme dilution. The final nucleosidetriphosphate substrate (BrdUTP) concentration was 16 μM he primer(odT₂₂) amount 12 ng per well. The RT reaction was allowed to proceedovernight (16-24 hours at 33° C. Thereafter the reaction was terminatedby a wash of the plate. The IC₅₀ value was defined as the concentrationof drug giving 50% inhibition of the RT activity studied.

[0054] Protocol for Isolation of Viral RT from Material which ContainsRT Blocking Antibodies, Based on Destruction of Soluble Cellular EnzymesFollowed by Isolation of Viral RT from Mini Columns.

[0055] 1) Label the 4.5 ml plastic tubes to be used. Place them in aNalgene box. Add 1 ml of sample (e.g. EDTA plasma from HIV infectedindividuals) to each labeled tube. Add 100 μl of a 66 mM solution of5,5′-dithiobis-(2-nitrobenzoic acid) in buffered water, vortex andincubate the samples for one hour at room temperature.

[0056] The activity of the free plasma enzymes is destroyed during thisprocedure while the enzymes contained within the virions remain intact.The virions can then be purified from 5,5′-dithiobis-(2-nitrobenzoicacid), enzyme activity blocking antibodies and other substances that mayinterfere with quantification of viral RT by several separationprocedures. The protocol below is based on the use of Fractogel® EMDTMAE Hicap gel.

[0057] 2) Suspend the separation gel carefully and transfer 1500 μl gelslurry to each sample pre-treatment tube.

[0058] 3) Incubate the samples with the gel slurry for 90 minutes atroom temperature with the tubes lying down horizontally on an orbitalshaker.

[0059] 4) Label the desired amount of 10 ml plastic mini columns toidentify the samples being analyzed. Mount the columns in a columnwashing device i.e. a Supelco Visiprep solid phase extraction vacuummanifold. Transfer the contents in the binding tubes to theircorresponding columns. Before transfer vortex the tube briefly to evenlydistribute the gel.

[0060] 5) When all the columns are filled, apply the vacuum and suck thegels dry. Turn off the vacuum and start the washing by filling eachcolumn with 9 ml buffer A. When all columns have been filled, apply thevacuum and suck the gels dry.

[0061] 6) Repeat step 5 three more times, giving a total of four washes.Suck the gels dry after each wash. After sucking the gels dry after thefourth wash, turn off the vacuum and proceed to step 7.

[0062]  The washing step removes unbound RT blocking antibodies and5,5′-dithiobis-(2-nitrobenzoic acid) from the system.

[0063] 7) Add to all dry gels 9 ml of conditioning buffer (B). After oneminute apply vacuum and suck the gels dry.

[0064] 8) Repeat step 7. Before turning off the vacuum check that allconditioning buffer (B) has been removed from all gels.

[0065] 9) Lift off the upper part of the column wash device. Mount thetube holder with the labeled tubes into a clean container. Refit theupper part of the device. Control that the small tubings from eachcolumn go down in their corresponding tubes.

[0066] 10) Add 600 μl lysis buffer (C) to each column. Let the bufferstand in the column for five minutes. Then apply the vacuum slowly andsuck the gels dry. This will in each tube give approximately 600 μl ofvirus lysate from the connected gel.

[0067] The RT activity recovered in the lysates from step 10 areessentially free from RT blocking antibodies, drugs and cellularpolymerase activity, and can be quantified with a sensitive RT activityassay, i.e. the Cavidi HS-kit Lenti RT, which is based on the methoddescribed by Ekstrand et al [7]. 25 μl lysate obtained according to thecurrent protocol is sufficient for determination of the RT activity inthe sample. The Remaining 575 μl sample should be frozen at −70° C.below for later use in the drug sensitivity test.

[0068] Note: RT enzymes that are not sensitive to cystein modifyingagents e.g. wild type HIV 1 RT can optionally be assayed in the presenceof up to 5 mM 5,5′-dithiobis-(2-nitrobenzoic acid). Sensitive enzymessuch as MULV RT and RT from certain therapy resistant HIV 1 strains(containing e.g. the mutation Y181C) on the other hand require additionof a sulfhydryl reducing agent i.e. cystein or cysteamine to the lysisbuffer.

[0069] Materials

[0070] Primer/Template for DNA Polymerase Assay.

[0071] The primer sequence is 18 bases 5′-GTC-CCT-GTT-CCG-GCG-CCA-3′(SEQID NO: 12) and linked at the 5′ end to a primary amine by a C6 spacerarm.

[0072] The template construct contains three parts with differentfunctions. From the 5′ end: A (A)n polymer used to amplify the BrdUsignal, a variable part (GTCA)m to obtain a polymerase reaction that isdependent on the four deoxynucleoside triphosphates and a sequencecomplementary to the primer.

[0073] In the experiments included in the examples n=12 and m=5, if nototherwise is stated.

[0074] Nucleosides, Enzyme Inhibitors and Antiviral Drugs

[0075] ddATP, 2′3′ dideoxyadenosine triphosphate; ddGTP, 2′3′dideoxyguanosine triphosphate;

[0076] ddCTP, 2′3′ dideoxy cytidine triphosphate; ddTTP, 2′3′ dideoxythymidine triphosphate.

[0077] TMAU, 6-([3,4-trimethylene]anilino)uracil

[0078] Tenofovir, (R)-9-(2-phosphonylmethoxy-propyl)adenine; Nevirapine,(11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2′,3′-f][1,4]diazepin-6-one)(NVP); and Efavirenz,(-)6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one)(EFV).

[0079] Enzymes

[0080] DNA polymerase I (E. coli) was purchased from AmershamBioscience. Recombinant DNA polymerase III from Staphylococus aureus wasproduced as described (Brown et al 1998). Mammalian DNA polymerase α(calf thymus) and β (human) were purchased from CHIMERx (Milwaukee). DNApolymerase γ was purified from beef heart as described (Pileur et al2000).

[0081] NNRTI resistant mutant forms of HIV-1 RT were produced (L100I,K103N, L100I/K103N, Y181C). As template for the mutations was used thepETRT expression vector, which was constructed from the BH10 isolate.Mutations were generated using commercial site-directed mutagenesiskits, QuikChange (Stratagene). The mutations were verified by DNAsequence analysis. The mutated and native forms of RT were isolated aspreviously described (Lindberg et al 2002).

[0082] The procedure for production of recombinant HIV-1 RTs with AZTspecific mutations were similar but the mutations were introduced intothe RT-coding region from the HXB2-D isolate.

[0083] Plasma Samples from HIV Infected Individuals.

[0084] Plasma samples from treatment naïve patients or from patientstreated with ordinary combination therapy were selected retrospectively.The amount of HIV-1 RNA in each sample was measured by standard HIV 1RNA PCR (Cobas, Roche Diagnostica). Serum samples from patients withlympho proliferative disorders were obtained from the Department ofInternal Medicine, Uppsala University, Akademiska sjukhuset, Uppsala

[0085] Separation gel: e.g. Fractogel® EMD TMAE or Fractogel® EMD TMAEHicap in 314 mM (2-(N-Morpholino)ethanesulfonic acid) (MES) pH 5.1, 413mM Potassium iodide and Heparin 0.5 mg/ml.

[0086] Mini columns, e.g. Biorad Poly-Prep® (7311553)

[0087] Mini column washing device, i.e. Supelco Visiprep solid phaseextraction vacuum manifold.

[0088] Plastic tubes, e.g. Nunc 4.5 ml cryogenic tubes.

[0089] Microtiter plates with immobilised prA, i.e. Nalge NuncNucleoLinck®

[0090] Cysteine modifying agent, e.g. 66 mM5,5′-dithiobis-(2-nitrobenzoic acid) in water buffered with 0.87 MTris(hydroxymethyl)aminomethane (pH 8.3).

[0091] Mild sulfhydryl reducing agent, e.g. 33 mM cysteamine in water.

[0092] Buffers Used:

[0093] A) Wash buffer: 20 mM MES pH 5.4, 500 mM Potassium acetate (KAc)

[0094] B) Conditioning buffer. An RT assay compatible buffer e.g. 50 mM(N-(2-Hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) (Hepes) pH 7.6,KAc 25 mM, magnesium chloride (MgCl₂) 20 mM,EthyleneGlycol-bis(β-aminoethyl Ether) N,N,N′,N′-Tetraacetic Acid (EGTA)0.2 mM, spermine 2 mM and heat inactivated bovine serum albumin (BSA)0.5 mg/ml.

[0095] C) Lysis buffer: An RT assay compatible buffer including adetergent e.g. 1.25% Polyoxyethylene 4 Lauryl Ether (Brij 30), 13 ng/mlodT₂₂ and the same components as in the conditioning buffer (B). Asulfhydryl reducing agent, i.e. 0.2 mM cysteamine is optionally addedwhen processing viruses with RT that are sensitive to SHoxidation/modification.

[0096] RT Reaction Mixture:

[0097] (N-(2-Hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) (Hepes)11.7 mM pH 7.6, BrdUTP 28.3 μM, odT₂₂ 120 ng/ml, MgCl₂ 4 mM, dextranesulphate 0.05 g/l, spermine 2 mM, Triton-X 100 0.5%(v/v),EthyleneGlycol-bis(β-aminoethyl Ether) N,N,N′,N′-Tetraacetic Acid (EGTA)0.2 mM and bovine serum albumin (BSA) 0.5 mg/ml.

[0098] DNA Polymerase γ and Retro DNA Polymerase Base Buffer.

[0099] Hepes 50 mM pH 8.0, MgCl₂ 8 mM, dextrane sulphate 1.5 μg/l,spermine 1 mM, Triton-X 100 0.5%(v/v), EGTA 0.2 mM, dithiothreitol (DTT)1.5 mM and bovine serum albumin (BSA) 0.5 mg/ml.

[0100] DNA Polymerase III Base Buffer.

[0101] (2-(N-Morpholino)ethanesulfonic acid) (MES) 40 mM pH 6.8,Potassium acetate (KAc) 40 mM, MgCl₂ 10 mM, spermine 2 mM,polyoxyethylenesorbitan monolaureate (Tween 20) 0.5%(v/v), EDTA 0.1 mM,dithiothreitol 1 mM and bovine serum albumin (BSA) 50 μg/ml.

[0102] DNA Polymerase β Base Buffer.

[0103]3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-1-propanesulfonic acid(AMPSO) 20 mM pH 8.3, MgCl₂ 1 mM, Spermidine 3 mM, BSA1 μg/ml, EDTA 10μM, DTT 0.1 mM, Tween 20 0.01%.

[0104] DNA Polymerase Reaction Mixture.

[0105] DNA polymerase base buffer reinforced with 24 μM BrdUTP, 49.5 μMdGTP, 49.5 μM dATP, 49.5 μM dCTP and 500 ng template/ml.

[0106] Retro DNA Polymerase Reaction Mixture with ATP.

[0107] The DNA polymerase γ and Retro DNA polymerase reaction mixturewas reinforced with 3.2 mM ATP and pH was adjusted to 7.1.

EXAMPLES Example 1 Utilization of Different Templates for Second StrandSynthesis by HIV RT

[0108] Two step dilution series of indicated template constructsstarting from 200 ng/ml were included in each well of mictrotiter plateswith immobilized primer according to “Production of primer coatedmicrotiter plates”. 100 fg recombinant HIV 1 RT was added to each welland the duration of the RT reaction was 18 hours. The polymeraseactivity on each template was determined according to “Protocol forDNA-polymerase assay” using DNA polymerase γ and Retro DNA polymerasebase buffer. 50 mM NaCl or alternatively 100 mM was used during thebinding of anti-BrdU monoclonal antibody. The activities found wereplotted towards concentration of template and the maximal signalachieved for each template type were calculated from the plateau valueof respective graph.

[0109] The results are summarized in Table 1. A clear correlation wasfound between the length of the A-tail and maximal signal obtainable inthe polymerase assay. The length of the A-tail did also affect theability of the antibody used for product detection to bind at increasedionic strength.

Example 2 Effects of Template Sequence on Inhibition of DNA PolymeraseIII with TMAU

[0110] 6-anilinouracils are selective inhibitors of DNA polymerase IIIfrom gram-positive bacteria. The anilinouracil molecule inhibits itspolymerase III target by sequestering it into an inactiveDNA-drug-protein complex (Tarantino et al 1990). The drug TMAU can beregarded as an analogue to GTP. The inhibitory capacity of indicatedconcentrations of TMAU towards 1.25 ng/well recombinant DNA polymeraseIII was determined according to “Protocol for determination ofinhibition of second strand synthesis on variable DNA template”utilizing either (CTGA)6-A12 (SEQ ID NO: 10) (▪) or (CTG)6-A3 (SEQ IDNO: 11) (▴) as template. The polymerase reaction time was one hour andthe GTP concentration in the DNA polymerase III reaction mixture wasreduced to 2.5 μM. The polymerase activities obtained on indicatedtemplate were recalculated into % of the activities found with the samepolymerase incubated in absence of inhibitor.

[0111] The results are depicted in FIG. 1. This highly specificinhibitor exhibited varying inhibitory capacity depending on thesequence of the template used. The current invention provides a systemwere the DNA template used easily can be changed to account for thespecific conditions required by the enzyme or inhibitor studied.

Example 3 Detection Sensitivity of the DNA Polymerase Assay

[0112] The activity of serial dilutions of HIV 1 wild type RT (♦),mammalian DNA polymerase β () and E. coli DNA polymerase I (▴) weremeasured according to “Protocol for DNA-polymerase assay” utilizing basebuffers optimized for indicated enzyme. The polymerase reaction timeused was over-night (18 hours). The results are depicted in FIG. 2. Eachof the three enzyme preparations displayed a linear relationship betweenamount of enzyme used and amount of product recovered. The assaybackground was 3800 rfu/hour. Utilizing double background as cut-offvalue for significant signal detection it was possible to detect down to1 nU HIV 1 wild type RT, 6 nU mamalian DNA polymerase β and 3 nU E. coliDNA polymerase I.

Example 4 The Activity of Five DNA Polymerases in Different AssaySystems

[0113] The activity of serial dilutions of DNA polymerase α, DNApolymerase β, DNA polymerase γ, serum from a patient suffering fromnon-Hodgkin's lymphoma and HIV-1 RT were measured according to “Protocolfor DNA-polymerase assay” and “Protocol for determination of RTactivity” using reaction solutions based on indicated polymerase basebuffers. The polymerase reaction time used was 2 hours. The activitiesfound were recalculated into % of the activity on variable DNA templateat the optimal reaction conditions for each enzyme. The results areshown in Table 2. Each of the five enzyme preparations investigated hadtheir individual preferences concerning optimal reaction conditions. DNApolymerase a and the human serum polymerase, however, displayed asimilar pattern. Only HIV-1 RT and DNA polymerase γ gave a significantactivity in the reverse transcriptase assay.

Example 5 Demonstration of the Ability of the DNA Polymerase Assay toMeasure Inhibition by the Dideoxy Analogues to All the Four DNA Bases

[0114] The inhibitory capacity of indicated concentrations of ddATP (▪),ddGTT (♦), ddCTP () and ddTTP (▴) towards the activity of 80 fgrecombinant wild type HIV 1 RT was determined according to “Protocol fordetermination of inhibition of second strand synthesis on variable DNAtemplate”. The polymerase activity at each inhibitor concentration wasrecalculated into % of the activity of a control in absence ofinhibitor. The results are depicted in FIG. 3.

[0115] The polymerase reaction utilizing the template (CTGA)₆-A₁₂ (SEQID NO: 10) was found sensitive to inhibition by the dideoxy analogues toall the four DNA bases. The IC₅₀ values found varied from 20 nM forddCTP to 80 nM for ddATP.

Example 6 Comparison of the Effect of Non-Nucleoside Inhibitors on Firstand Second Strand DNA Synthesis by HIV 1 RT

[0116] The effects of three non-nucleoside inhibitors on indicatedrecombinant HIV 1 RT were determined according to “Protocol fordetermination of inhibition of second strand synthesis on variable DNAtemplate” using DNA polymerase γ and Retro DNA polymerase base bufferand “Protocol for determination of inhibition of reverse transcription ”respectively. The duration of the polymerization reactions were 19 hoursfor the second strand synthesis on (CTGA)₆-A₁₂ (SEQ ID NO: 10) and 2hours for the first strand synthesis on prA respectively. The RTactivities obtained were recalculated into % of the activities foundwith the same RT incubated in absence of inhibitor.

[0117] The results are summarized in Table 3. Both assay systems had thecapacity to distinguish between resistant (Y181C, V179D) and sensitiveRT enzymes. The IC50 values for any of the inhibitors was notsignificantly affected of a five fold difference in the amount of enzymeused in any of the two assay systems. Further there was no significantdifference between the IC50 values achieved by measuring inhibition offirst or second strand DNA synthesis.

Example 7 Demonstration of the Biochemical Mechanism UnderlyingResistance to the Antiviral Drug Tenofovir

[0118] Prolonged therapy of HIV infected individuals with nucleosideanalogues leads to the development of resistant virus. This process isassociated with the gradual appearance of mutations in the viral polgene. The effects of these substitutions at the enzymatic levels arecomplicated and include enhancement of a primitive DNA editing function.This reaction is nucleotide dependent and produces dinucleosidepolyphosphate and an extendible DNA 3′ end.

[0119] The effects of serial dilutions of tenofovir triphosphate on 4pg/well of indicated recombinant HIV 1 RT were determined according to“Protocol for determination of inhibition of second strand synthesis onvariable DNA template” utilizing standard reaction solution based on DNApolymerase γ and Retro DNA polymerase base buffer and the same reactionsolution supplemented with ATP. The duration of the polymerizationreactions was 19 hours. The RT activities obtained were recalculatedinto % of the activity of the same RT incubated in absence of inhibitor.FIG. 4 shows the resuts.

[0120] Symbols: HIV-1 wild type RT in standard DNA polymerase reactionsolution (◯). HIV-1 wild type RT in DNA polymerase reaction solutionwith ATP (), T69S→SS/L210W/T215Y HIV-1 mutant RT in standard DNApolymerase reaction solution (⋄),

[0121] T69S→SS/L210W/T215Y HIV-1 mutant RT in DNA polymerase reactionsolution with ATP(♦).

[0122] The results depicted in FIG. 4 demonstrate that the difference indrug susceptibility between wild type and mutant RT increasedapproximately 10 fold when a reaction solution with capacity to supportan ATP dependent phosphorolysis reaction was used.

Example 8 Determination of the Susceptibility to Nevirapine Using PlasmaDerived RT

[0123] One ml samples of plasma from 3 HIV infected individuals fromStockholm, Sweden were processed according to “Protocol for isolation ofviral RT from material which contains RT blocking antibodies, based ondestruction of soluble cellular enzymes followed by isolation of viralRT from mini columns.” Each plasma RT and two control enzymes weretitrated towards a set of serial dilutions of Nevirapine according to“Protocol for determination of inhibition of second strand synthesis onvariable DNA template” using “DNA polymerase γ and Retro DNA polymerasebase buffer”. See FIG. 5.

[0124] Symbols: (□) RT from patient 1 having 140 000 genome copies/ml(▪)RT from patient 2 having 180 000 genome copies/ml, (▴) RT frompatient 3 having 390 000 genome copies/ml, () a control consisting ofrecombinant HIV-1 wild type RT, (♦) and a control consisting of therecombinant HIV-1 mutant RT (L100I), with intermediate Nevirapineresistance.

[0125] The IC50 values towards Nevirapine found for the patient RTsvaried from 0.7 to 1.2 μM. To be compared with 0.5 μM for the controlwild type RT and >10 μM for the mutant RT with intermediate Nevirapineresistance (L100I).

Example 9 Detection of a DNA Polymerase Activity in Serum from Patientswith Lymphoproliferative Disorders

[0126] Serum from four patients suffering from non-Hodgkin's lymphomaand from six healthy blood donors were serially diluted in DNApolymerase III base buffer. The polymerase activity at each dilutionstep were measured according to “Protocol for DNA-polymerase assay”using two and six hours polymerase reaction time.

[0127] The DNA polymerase activity/μl serum sample and hour polymeraseassay was calculated at the dilution range were there was a linearrelationship between the amount of product formed and the amount ofplasma added to the assay (see table 4).

[0128] Each serum sample from the patients with non-Hodgkin's lymphomacontained individual amounts of DNA polymerase activity, with similarproperties as DNA-polymerase α (see table 3). The amount of activityranged from approximately 2 to 190 times the average value found amonghealthy blood donors. TABLE 1 Utilization of different templates forsecond strand synthesis by HIV RT. Signal Back- at 100 mM Maximal groundNaCl^(a) (% of signal* (rfu/ maximal Template used (rfu/hour) hour)signal) SEQ ID NO: (CTGA)5 89444 1629 20 SEQ ID NO: 1 (CTGA)5-A 636941958 25 SEQ ID NO: 2 (CTGA)5-AA 184421 613 33 SEQ ID NO: 3 (CTGA)5-AAAA239688 2565 44 SEQ ID NO: 4 (CTGA)5-A8 198894 1544 58 SEQ ID NO: 5(CTGA)6 60627 1285 22 SEQ ID NO: 6 (CTGA)6-AAA 83555 1009 ND SEQ ID NO:7 (CTGA)6-A5 119326 914 ND SEQ ID NO: 8 (CTGA)6-A9 202668 963 ND SEQ IDNO: 9 (CTGA)6-A12 226062 726 ND  SEQ ID NO: 10 (CTG)6-A3 124334 814 ND SEQ ID NO: 11

[0129] TABLE 2 Apparent activity of four mammalian DNA polymerases andHIV RT in different assay systems Activity of DNA polymerase isozyme inindicated assay (% *) DNA DNA DNA serum Base buffer pol α pol β pol γDNAp HIV RT Pol III 100 17 0 100 0 DNA pol β 40 100 5 30 33 DNA polγ^(a) 1 13 100 9 100 prA/Lenti^(b) 0 0 10 0 2175

[0130] TABLE 3 Comparison of the effect of non-nucleoside inhibitors onfirst and second strand DNA synthesis by HIV 1 RT. RT enzyme IC₅₀ (μm)on template* Inhibitor Type amount (fg) (CTGA)₆-A₁₂ prA Nevirapine wt100 2.0 2.2 wt 20 2.0 2.5 Y181C 100 >500 200 Y181C 20 >500 180 V179D 1008 7 V179D 20 12 8 Efavirenz wt 100 0.04 0.02 wt 20 0.03 0.02 Y181C 1000.12 0.15 Y181C 20 0.14 0.13 V179D 100 0.5 0.7 V179D 20 0.4 0.6Foscarnet wt 100 0.5 0.7 wt 20 0.5 0.6 # the second strand synthesis on(CTGA)₆-A₁₂ (SEQ ID NO: 10) and 2 hours for the first strand synthesison prA respectively. The RT activities obtained were recalculated into %of the activities found with the same RT incubated in absence ofinhibitor.

[0131] TABLE 4 Detection of a DNA polymerase activity in serum frompatients with lymphoproliferative disorders. Polymerase activity(rfu/hour/μl serum/ Serum code Serum origin hour polymerase reaction))T1 NHL patient^(a) 158521 T2 NHL patient 1305 T3 NHL patient 3405 T4 NHLpatient 16619 B1-B6 Blood donors 841 ± 180

[0132] Serum from four patients suffering from non-Hodgkin's lymphomaand from six healthy blood donors were serially diluted in DNApolymerase III base buffer. The polymerase activity at each dilutionstep were measured according to “Protocol for DNA-polymerase assay”. TheDNA polymerase activities tabulated was calculated from the dilutionrange were there was a linear relationship between the amount of productformed and the amount of plasma added. The assay background 854 flu/hhas been subtracted from all values.

[0133] References

[0134] Aposhian H V, Kornberg A. J.Biol.Chem. 1962, 237, 519

[0135] Arion D, Kaushik N, McCormick S, Borkow G, Parniak M A.Phenotypic mechanism of HIV-1 resistance to 3′-azido-3′-deoxythymidine(AZT): increased polymerization processivity and enhanced sensitivity topyrophosphate of the mutant viral reverse transcriptase. Biochemistry.1998 Nov. 10;37(45):15908-17.

[0136] Barnes M H, Leo C J, Brown N C. DNA polymerase III ofGram-positive eubacteria is a zinc metalloprotein conserving anessential finger-like domain. Biochemistry. 1998 Nov. 3;37(44): 15254-60

[0137] Eberle J, Seibl R, Kessler, C, Konig, Bernhard B. Reagents andkits for determining polymerase activity. 1997 U.S. Pat. No. 5,635,350:

[0138] Ekstrand D H, Awad R J, Källander C F, Gronowitz J S. A sensitiveassay for the quantification of reverse transcriptase activity based onthe use of carrier-bound template and non-radioactive-product detection,with special reference to human-immunodeficiency-virus isolation.Biotechnol Appl Biochem. 1996 Apr.; 23 (Pt 2):95-105.

[0139] Goff, S. P. (1990) Retrovirus reverse transcriptase: Synthesis,Structure, and Function. Review. J Acquir Imm Defic Syndr 3: 817-831.

[0140] Gronowitz; J S, Källander, C F R Method of determining dTkisoenzyme activity and the use thereof. 1987 U.S. Pat. No. 4,637.977:

[0141] Meyer P R, Matsuura S E, Mian A M, So A G, Scott W A. RelatedArticles. A mechanism of AZT resistance: an increase innucleotide-dependent primer unblocking by mutant HIV-1 reversetranscriptase. Mol Cell. 1999 July;4(1):35-43

[0142] Pileur F, Toulme J J, Cazenave C. Eukaryotic ribonucleases HI andHII generate characteristic hydrolytic patterns on DNA-RNA hybrids:further evidence that mitochondrial RNase H is an RNase HII. NucleicAcids Res. 2000 Sep. 15;28(18):3674-83

[0143] Seville M, West A B, Cull M G, McHenry C S. Fluorometric assayfor DNA polymerases and reverse transcriptase. Biotechniques. 1996October;21(4):664, 666, 668, 670, 672.

[0144] Tarantino P M Jr, Zhi C, Wright G E, Brown N C. RelatedInhibitors of DNA polymerase III as novel antimicrobial agents againstgram-positive eubacteria. Antimicrob Agents Chemother. 1999August;43(8):1982-7.

[0145] Tveit H, Kristensen T. Fluorescence-based DNA polymerase assay.Anal Biochem. 2001 Feb. 1;289(1):96-8.

[0146] Vandamme A M, Van Vaerenbergh K, De Clercq E. Anti-humanimmunodeficiency virus drug combination strategies. Antivir ChemChemother. 1998 May;9(3):187-203.

1 12 1 20 DNA Artificial Sequence Description of Artificial Sequencetemplate 1 ctgactgact gactgactga 20 2 21 DNA Artificial SequenceDescription of Artificial Sequence template 2 ctgactgact gactgactga a 213 22 DNA Artificial Sequence Description of Artificial Sequence template3 ctgactgact gactgactga aa 22 4 24 DNA Artificial Sequence Descriptionof Artificial Sequence template 4 ctgactgact gactgactga aaaa 24 5 28 DNAArtificial Sequence Description of Artificial Sequence template 5ctgactgact gactgactga aaaaaaaa 28 6 24 DNA Artificial SequenceDescription of Artificial Sequence template 6 ctgactgact gactgactga ctga24 7 27 DNA Artificial Sequence Description of Artificial Sequencetemplate 7 ctgactgact gactgactga ctgaaaa 27 8 29 DNA Artificial SequenceDescription of Artificial Sequence template 8 ctgactgact gactgactgactgaaaaaa 29 9 33 DNA Artificial Sequence Description of ArtificialSequence template 9 ctgactgact gactgactga ctgaaaaaaa aaa 33 10 36 DNAArtificial Sequence Description of Artificial Sequence template 10ctgactgact gactgactga ctgaaaaaaa aaaaaa 36 11 21 DNA Artificial SequenceDescription of Artificial Sequence template 11 ctgctgctgc tgctgctgaa a21 12 18 DNA Artificial Sequence Description of Artificial Sequenceprimer 12 gtccctgttc cggcgcca 18

1. A method for measuring DNA-dependent DNA polymerisation in abiological sample, comprising the steps of a) providing a primer with asingle stranded short specific sequence, which is unable to base pairinternally, bound to a solid phase, b) contacting the primer constructwith a reaction mixture containing a single stranded deoxynucleotidetemplate with a part of the sequence complementary to the primer and thefour deoxynucleoside triphosphates, one of which is modified so that itis specifically recognized by a labeled antibody, c) adding a biologicalsample comprising the DNA polymerase to the mixture of b), d) allowingthe polymerase reaction to proceed, e) incubating the immobilizedreaction product resulting from d) with the labeled antibody, f)detecting the amount of bound labeled antibody with the aid of the labelused, and g) measuring the amount of incorporated modifieddeoxynucleoside triphosphate, as a measure of the DNA polymerization,with the aid of the label of the bound antibody.
 2. The method accordingto claim 1, wherein the DNA polymerase is a retrovirus reversetranscriptase (RT).
 3. The method according to claim 2, wherein theretrovirus RT is human immunodeficiency virus (HIV) RT.
 4. The methodaccording to any one of claims 1-3, wherein the modified deoxynucleosidetriphosphate is 5-bromodeoxyuridine 5′-triphosphate (BrdUTP) and thelabeled antibody is an alkaline phosphatase (Ap) conjugated anti-BrUmonoclonal antibody.
 5. The method according to any one of claims 1-4,wherein the measured DNA polymerisation is used for drug susceptibilitytesting.
 6. Commercial package containing written and/or data carrierinstructions for measuring DNA-dependent DNA polymerisation according toany one or claims 1-4, comprising a) a primer with a single strandedshort specific sequence, which is unable to base pair internally, boundto a solid phase, b) a single stranded deoxynucleotide template with apart of the sequence complementary to the primer in a), c) the fourdeoxynucleoside triphosphates, one of which is modified so that it isspecifically recognized by a labeled antibody, and d) the labelledantibody that recognizes the modified deoxynucleoside triphosphate inc).